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Application of substance capable of reducing expression of zinc finger protein CTCF to preparation of drugs for treating leukemia

A technology of zinc finger protein and expression cassette, which is applied to the application field of substances that reduce the expression of zinc finger protein CTCF in the preparation of leukemia drugs, and can solve the problems of no obvious improvement in the cure rate of ALL and unclear pathogenesis of leukemia, so as to promote the occurrence of leukemia. Apoptosis, inhibition of apoptosis, effect of increasing sensitivity

Inactive Publication Date: 2013-08-07
BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the 1980s, due to multi-drug combination intensive chemotherapy and strong supportive treatment, the cure rate of ALL in children has reached about 80%, but the cure rate of ALL has not improved significantly in the past 30 years, mainly because the pathogenesis of leukemia is still unknown

Method used

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  • Application of substance capable of reducing expression of zinc finger protein CTCF to preparation of drugs for treating leukemia
  • Application of substance capable of reducing expression of zinc finger protein CTCF to preparation of drugs for treating leukemia
  • Application of substance capable of reducing expression of zinc finger protein CTCF to preparation of drugs for treating leukemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1, RNA interference recombinant expression vector construction of zinc finger protein CTCF

[0053] 1. Selection of RNA interference target sequence

[0054] For the full-length cDNA sequence (SEQ ID No.6) of the zinc finger protein CTCF coding gene CTCF, the following three DNA sequences were selected as target sequences for RNA interference:

[0055] sh-1: Positions 809-827 of SEQ ID No.6 (ie 5'-TGACTGTACCTGTTGCTAC-3')

[0056] sh-2: 1103-1122 of SEQ ID No.6 (ie 5'-ATGTAGATGTGTCTGTCTAC-3')

[0057] sh-3: No. 1398-1416 of SEQ ID No.6 (ie 5'-TACTCGTCCTCACAAGTGC-3')

[0058] Positions 445-2628 in SEQ ID No.6 are open reading frames encoding the zinc finger protein CTCF shown in SEQ ID No.7.

[0059] 2. Small interfering RNA (siRNA)

[0060] Three siRNAs (siRNA-1, siRNA-2, and siRNA-3) were designed against the three target sequences of the zinc finger protein CTCF in step 1, respectively. The target sequence of siRNA-1 is sh-1, the target sequence of siRNA-2...

Embodiment 2

[0113] Example 2, RNAi gene knockout recombinant vector transfection of leukemia cells

[0114] 1. Cell culture for transfection

[0115] 24 hours before transfection, use RPMI-1640 medium containing 10% fetal bovine serum (FBS) in a 15ml culture dish at 37°C and 5% CO 2 The B-ALL cell line NALM-6 was cultured under the conditions and transfected when it reached 75%-90%.

[0116] 2. Transfection

[0117] The RNA interference recombinant expression vectors pDsU6-GFP-sh-1, pDsU6-GFP-sh-2, pDsU6-GFP-sh-3 and the vector pDsU6-GFP-sh-luc (RNAi Positive control, for silencing luciferase gene) respectively transfected the cells cultured in step 1 to obtain the recombinant leukemia cells NALM-6 / pDsU6-GFP-sh-1 containing RNA interference recombinant expression vector pDsU6-GFP-sh-1, Recombinant leukemia cell NALM-6 / pDsU6-GFP-sh-2 containing RNA interference recombinant expression vector pDsU6-GFP-sh-2, recombinant leukemia cell NALM-6 / pDsU6-GFP-sh-3 containing RNA interference recom...

Embodiment 3

[0120] Example 3, Western Blot Detection of Expression of Zinc Finger Protein CTCF in Recombinant Leukemic Cells

[0121] Take four kinds of recombinant leukemia cells NALM-6 / pDsU6-GFP-sh-1, NALM-6 / pDsU6-GFP-sh-2, NALM-6 / pDsU6-GFP-sh-3 after transfection 72h in Example 2 and NALM-6 / pDsU6-GFP-sh-luc, the total protein was extracted respectively, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal reference for Western blot detection. The primary antibody for detecting zinc finger protein CTCF was CTCF( Molecular weight 83KDa) monoclonal antibody (purchased from Millipore), the primary antibody for detecting internal reference GAPDH is GAPDH (molecular weight 34KDa) monoclonal antibody (purchased from Shanghai Kangcheng Company in China), the results are as follows figure 1 As shown, the results show that: the recombinant leukemia cell NALM-6 / pDsU6-GFP-sh-1 containing RNA interference recombinant expression vector pDsU6-GFP-sh-1, and the recombinant expr...

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Abstract

The invention discloses application of a substance capable of reducing expression of a zinc finger protein CTCF to preparation of drugs for treating leukemia. In particular, the substance reducing expression of the zinc finger protein CTCF can be short hairpin RNA (shRNA) forming a stem-loop structure. The name of the shRNA is shRNA-3. One chain sequence of the stem in the stem-loop structure is SEQ ID No.1 and the other chain sequence of the stem in the stem-loop structure is SEQ ID No.2. The invention proves that the zinc finger protein CTCF is an anti-apoptosis factor and has the function of suppressing cell apoptosis in the ALL (acute lymphoblastic leukemia) cells. Apoptosis of tumor cells-leukemia cells can be promoted by interfering with expression of CTCF. The invention provides a new way for leukemia treatment. The zinc finger protein CTCF is expected to become a potential target for anti-leukemia treatment and has very extensive application prospect in the medical field.

Description

technical field [0001] The invention relates to the application of a substance for reducing the expression of zinc finger protein CTCF in the preparation of medicines for treating leukemia. Background technique [0002] Zinc finger protein CTCF, also known as CCCTC-binding factor, is a highly conserved multi-zinc finger structural protein consisting of 727 amino acids. It participates in a variety of biological regulatory functions, including transcriptional activation or repression, chromatin insulation, gene imprinting, and X chromosome inactivation. [0003] Studies have found that CTCF is involved in the occurrence and development of various human tumors. Tumor-specific mutations in the CTCF zinc finger structure have been found in breast cancer, prostate cancer, and Wilms tumor. This mutation causes CTCF to lose its ability to bind to downstream tumor target genes, resulting in abnormal expression of these genes, thereby initiating tumors. In addition, CTCF may parti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N1/21C12N1/15C12N1/13C12N5/10A61K48/00A61P35/02
Inventor 郑胡镛鲍时来张寒朱琳刘潇
Owner BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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